Intravital imaging has become a vital tool to study cells in their native context and yet there are almost no methods or tools suitable for real-time spatially-resolved analysis of signaling in living tissues. Light-based fluorescence has the best resolution for subcellular resolution of signaling and can be used in real-time. Numerous groups have developed protocols for applying multiphoton fluorescence imaging to target tissues and organs in the mouse. These protocols are currently limited to observation of cell morphology, positioning and motility parameters while progress requires development of probe systems that respond to signaling onset. In vitro, the use of GFP-tagged biomarkers, genetically expressed and used to track molecular behaviors within cells has proven to be extremely useful in tracking cellular activation. However, the situation in vivo has been considered difficult, as a result of considerations of probe intensity, difficulty with interpretation in the absence of fiducials, and regional autofluorescence. We show that, with new improved GaAs detection in 2-photon microscopy, excitation and detection of weak fluorescent protein expression is not the primary hindrance to molecular imaging so long as the molecule of interest is expressed at or near a now-defined level. Instead, the limitations are autofluorescence reduction and strategies that provide fiducial markers to characterize biosensing fusion-proteins. We propose that a collection of multiplexed biosensor arrays will permit the imaging of nuclear import of transcription factors, activation of PI3kinase enzymes and T cell receptors. The genes encoding these will be integrated as P2A-based fusion into the tissue-independent ROSA-26 locus with lox-stop- lox control so that they can be conditionally expressed under the many existing tissue-specific- promoter Cre driver-strains. Based on the design, which mimics our previous success with the T cell receptor first in vitro and now in vivo, these will be functional even in an organ with uneven levels of autofluorescence. Our goal is to develop these tools and subsequently apply them to a T cell interacting with a newly improved model for spontaneous tumor outgrowth. We will also make the mice expressing these biosensors available to the community through non-restrictive sharing and/or the placement of mouse strains into a repository.7. Project Narrative: While our science is achieving great success at understanding the behavior of our cells in isolation, it is more difficult to study the way cells work in their native context. This proposal will create new mouse strains that will permit us to look into tissues using microscopy and observe cells being activated to divide and/or remodel the tissue of which they are a part. It is important for us to understand which cells are being activated and deactivated in the context of many diseases of humans, and our results are likely to provide great insight into the nature of many of these.